Rumen bacteria plays an important role in digesting feed. The respective bacteria are adapted to low quality forage from agricultural by-product, especially those originated from rumen fluid of buffaloes. Study of innoculating buffalo rumen bacteria as probiotic into the rumen of Holstein Frisian calves have not been investigated so far. The aims of the present study were: (1) to isolate and characterize bacteria from buffalo, and (2) to evaluate the quality of isolates for enhancing forage digestion in Frisian Holstein calves. The study consisted of three studies: (1) Isolation, characterization, and genetic identification of buffalo rumen bacteria, (2) in vitro evaluation on various sources of forage and concentrates, and (3) in vivo evaluation into calves with different inoculation period. Design of the first study was based on a completely randomized factorial design with two factors: the type of bacteria and types of forage source, in which the diversity of bacteria were analyzed using NTSys 2.10 program. The second study was also based on a completely randomized factorial design, where the factors were various bacterial sources and feed types. The third study was consisted of two experiments, both were using completely randomized designs. The first experiment was to determine the effectiveness of bacteria consortium innoculation into Frisian Holstein calves with three calves received the respective inoculation (treatment group) and four calves without any inoculation (control group). The second experiment was todetermine the potency of bacteria consortium innoculation into Frisian Holstein calves during preweaning with three calves as treatment group and three calves as control. The results showed that: (1) buffalo rumen bacteria can grow well on forage substrates in which 14 isolates of bacteria possessed a high adaptability to fiber (high CMCase activity); these isolates could be clustered into six types which have similarity ≥ 51%. (2) Dry matter digestibility of concentrate were almost the same between the consortium of bacteria and buffalo rumen fluid. (3) buffalo rumen bacteria is a potential probiotic and could effectively be innoculated since preweaning period. (4) Inoculation of bacteria consortium increased feed intake and cobalt uptake on weaning period. (5) Inoculation of bacteria consortium did not cause any negative effects on ADG, physiological status and rumen fermentability.